Cancer is the second mortality cause in developed countries, with more than 11 millions people diagnosed as suffering from cancer each year, and 7 millions people killed by cancer each year in the world according to World Health Organization (WHO). Cancer is thus an important public health problem in developed countries, and the ageing of their population will cause these numbers to continue to increase even if age-specific rates remain constant.
In Europe in 2004, the most common incident form of cancer was lung cancer (13.3% of all incident cases), followed by colorectal cancer (13.2%) and breast cancer (13%). Lung cancer was also the most common cause of cancer death (341 800 deaths), followed by colorectal (203 700), stomach (137 900) and breast (129 900). To make great progress quickly against cancer, the need is evident to make a concerted attack on the big killers: lung, colorectal, breast and stomach cancer.
Conventional treatments against cancer include surgery, radiotherapy and chemotherapy. While surgery usually does not have many deleterious effects, it is not always possible and it is also usually not sufficient to cure cancer, since tumor cells may have escaped surgical removal. Thus, radiotherapy and chemotherapy are necessary, even when surgery is possible.
However, while most drugs may have deleterious effects, anticancer drugs are among those resulting in the worse adverse effects. Indeed, anticancer drugs are usually cytotoxic active agents with some preference for tumor cells, but which also display toxicity on other cells due to insufficient specificity for tumor cells, thus resulting in often serious adverse reactions. Although radiotherapy is more localized, it is also not specific of tumor cells and thus also results in serious adverse effects on healthy surrounding cells. As a result, while these treatments can be used during limited time periods, they cannot be continued after the tumor appears cured, and cancer relapses are often observed.
In addition, some patients develop resistance to their chemotherapy treatment.
There is thus a very important need for alternative cancer treatments with increased specificity for tumor cells and thus decreased adverse effects, and which might thus be used in alone or in combination with chemotherapy or radiotherapy and might in some cases (for instance when relapse is at high risk) be used in a more continuous therapy.
Among new anticancer molecules, monoclonal antibodies have emerged as a new class of successful drugs for hematopoietic malignant neoplasms and solid tumors; being now a major therapeutic strategy in clinical oncology.
Monoclonal antibodies are typically of the IgG class and generally react with antigens expressed on tumor target cells, with high specificity and affinity. They interfere with the functioning of cancer cells inactivating the targeted pathways, or bind to tumor cells and activate various cytotoxic mechanisms via recruitment of immune effectors (naked antibodies), or destroy cancer cells by focused delivery of radioisotopes or cellular toxins coupled to the IgGs (conjugated antibodies).
Since monoclonal antibodies are specific for a particular antigen, they will not affect cells that do not express their particular antigen. As a result, by selecting target antigens that are specifically expressed by tumor cells or that are implicated in mechanisms specifically needed by tumor cells, they display tumor specificity and thus generate much less adverse effects than conventional anticancer molecules.
Currently there are 9 naked or conjugated antibodies approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) for human cancer treatments. In addition, numerous antibodies are in late clinical trial phases.
The most significant recent advances in the application of monoclonal antibodies (mAbs) to oncology have been the introduction and approval of bevacizumab (Avastin), an anti-vascular endothelial growth factor antibody, and of cetuximab (Erbitux), an anti-epidermal growth factor antibody. In combination with standard chemotherapy regimens, bevacizumab significantly prolongs the survival of patients with metastatic cancer of the colorectum, breast and lung. Cetuximab, used alone or with salvage chemotherapy, produces clinically meaningful anti-tumor responses in patients with chemotherapy-refractory cancers of the colon and rectum. In addition, the anti-HER/neu antibody trastuzumab (Herceptin), in combination with standard adjuvant chemotherapy, has been shown to reduce relapses and prolong disease-free and overall survival in high-risk patients after definitive local therapy for breast cancer. These exciting recent results highlight the great potential of monoclonal antibodies therapies for cancer treatment and provide optimism for the development of mAbs that bind novel targets, exploit novel mechanisms of action or possess improved tumor targeting.
Current developed anticancer monoclonal antibodies mostly target tumor specific antigens. While this permits high tumor specificity and thus low toxicity, the drawbacks are that it limits the treatment to tumors expressing this particular antigen. As a result, not all tumor types may be treated, and even among a particular tumor type, some patient may not respond to the treatment if their tumor does not express the targeted antigen.
Another approach for the development of anticancer monoclonal antibodies is thus to target not the tumor itself but its microenvironment, and particularly molecules of the microenvironment that are necessary for tumor growth.
Epithelial tissue represents the major site for human carcinogenesis and is involved in more than 90% of all cancers. While numerous genetic anomalies have been identified in many of these epithelial tumors, important questions remain regarding the molecular mechanisms involved in malignant transformation of tumor cells and in the metastatic process.
In order for tumors to grow and metastasize, neoplastic cells must invade and migrate into surrounding tissues. Metastatic spread of tumors to distal organs is the primary cause of death of cancer patients. Metastasis is a complex process, which involves the coordination of several signal transduction pathways that allow cancer cells to proliferate, remodel their surrounding environment, invade and migrate through new tissues.
Cellular invasive and migratory behaviour is governed at both extracellular and intracellular levels and depends on the carefully balanced dynamic interaction of the cell with its extracellular matrix (ECM). It is well established that adhesion to ECM proteins plays a key role in the assembly of cells into functional multicellular organisms and that such adhesions are involved in transmembrane signalling processes that regulate cell behaviour and fate. Novel insights into the mechanisms that regulate cell survival as well as cell migration and invasion have led to the conclusion that these highly regulated mechanisms are altered in cancer cells. The nature of the cell-matrix adhesion proteins (or related to adhesion) which are specifically involved in tumor development and cell invasion is still a pending question.
The ability to block cell adhesion proteins involved in the migratory and invasive capacity of tumor cells offers a promising approach to the treatment of patients with malignant disease.
While integrins are the major cell surface receptors for the ECM, other adhesive systems have also been described, including matrix and transmembrane receptors carrying glycosaminoglycans (GAGs) molecules on their ectodomain such as syndecans.
Both the expression pattern of these ECM receptors and the ligand-binding affinity determine the adhesive properties of the cell. The ligand-binding affinity of integrins may be regulated by intracellular cues, but through extracellular interactions integrins are also capable of signalling to the cellular machinery and thereby affecting gene expression and several cellular functions. Integrins and syndecans, which lack enzymatic activity, transmit their intracellular signals by interacting with various effector proteins, including both structural and signalling molecules. Following ligand binding, they cluster in adhesion complexes that contain several different actin-associated proteins that coordinate the “integration” of ECM components with the cellular cytoskeletal machinery. Cell-matrix adhesion receptors also activate kinases and phosphatases that phosphorylate or dephosphorylate cytoskeletal proteins, and thus regulate stress fibre formation, cell shape and motility. Furthermore, the cytoplasmic tail of adhesion receptors can recruit signalling proteins that modulate cell adhesion to the ECM. Cell movement results from a dynamic remodeling of the actin cytoskeleton and of its interaction with the plasma membrane and adhesion receptors.
Laminins (LN) belong to a growing family of heterotrimeric proteins, commonly found in basement membranes (BMs). These large molecules promote cell adhesion and migration via integrins and proteoglycan-type receptors. Over 15 LN isoforms are known with variable cell- and tissue-specific expression and are differentially recognized by cellular receptors. Expression of LN isoforms in tumors usually reflects expression in their normal counterpart. However, loss of the BM barrier often occurs during tumor invasion. In carcinomas, tumor cells at the invading front strongly express the LN5 isoform, remodelling of the vascular BM is observed during angiogenesis, and penetration of several BMs occurs during tumor dissemination and metastasis. Thus, deregulated cell-LN interactions are major traits of malignant disorders.
Laminin 5 (LN5, also abbreviated as LN332) is a major component of epithelial BMs and is composed of 3 chains (alpha3, beta3, gamma2) assembled into a cross-shaped heterotrimer (Rousselle et al., 1991, see FIG. 1).
Previously published research and literature has demonstrated and suggested that certain regions of LN5 may provide suitable targets for antibodies having anti-cancer properties. WO 2005/056598, WO 2005/040219, and WO 2005/052003, for example, indicate that antibodies directed to the gamma2 chain of LN5, particularly domain 3 thereof, may be useful anti-cancer agents.
Previously published literature also suggests that the alpha3 chain of LN5 may similarly be a useful target for anti-cancer antibodies. Various monoclonal antibodies directed to human LN5 (hLN5) alpha3 chain have thus been described, some of them being presented as useful for treating cancer.
The alpha3 chain comprises a large globular domain in its carboxyl-terminal region (G domain), which consists of five homologous globular subdomains, each containing about 200 amino acids (LG1-LG5). LN5 alpha3 chain canonical sequence is defined under UniProtKB/Swiss-Prot accession number Q16787-1, as represented by SEQ ID NO:1 (isoform A). Another isoform (isoform B) with incomplete sequence, as represented by SEQ ID NO:2 (UniProtKB/Swiss-Prot accession number Q16787-2), has been identified. Positions of the G1 to G5 domains in SEQ ID NO:1 (canonical isoform A) are displayed in following Table 1:
TABLE 1Position of domains G1 to G5 in LN5 alpha3 chainDomainPositionLengthG1770-971202G2 978-1140163G31147-1307161G41366-1530165G51537-1710174
After secretion and deposition of precursor LN5 (pre-LN5) in the BM, one event results in the cleavage of the globular domains 4 and 5 (LG4/5) of the alpha3 chain. The resulting mature LN5 is the major component of anchoring filaments in skin, where it mediates cell adhesion via interaction of the LG1-3 triplet domain with both alpha3beta1and alpha6beta4integrins (Carter et al., 1991; Rousselle and Aumailley, 1994).
Antibodies to LN5 alpha3 chain may thus be directed either not to the G domain or, within the G domain, either to the LG1-LG3 subdomains that are present both in pre-LN5 and in mature LN5, or in the cleaved LG4-LG5 domains.
The 5 LG subdomains contains multiple cell binding sites with different mechanisms and different functions and are thus considered relevant to a number of cellular functions associated with LN5.
WO 2000/26342 suggests the use of various specific antibodies against LN5 alpha3 globular domains, generally, including monoclonal antibodies to human LN5 alpha3 chain with an observed effect on LN5 function named BM165, CM6, RG13 and P3H9-2, although no specific features of these antibodies are described. However, some of these antibodies have been further studied and characterized.
BM165 has been further shown to be directed against LN5 alpha3 LG2 domain and compared to antibody 7B2 also directed to LN5 alpha3 LG2 domain, which is suggested to inhibit migration and adhesion and to display antitumor activity (WO2008/005828).
RG13 has also been shown to be directed against LN5 alpha3 LG2 domain and to block migration and proliferation (Gonzales et al, 1999 and Goldfinger et al., 1999).
CM6 recognizes both pre-LN5 and mature LN5, thus demonstrating that the epitope is not situated in LG4-LG5 domains. It was shown to block adhesion and proliferation of tumor cells, without inducing apoptosis (Baker et al, 1996; Gonzales et al, 1999).
P3H9-2 recognizes a binding site for integrin and has been shown to inhibit adhesion and proliferation (Gonzales et al, 1999; Kim et al., 2000; and Wayner et al., 1993).
As indicated above, the LG1-3 triplet domain has been reported to interact with both alpha3beta1 and alpha6beta4 integrins (Carter et al., 1991; Rousselle and Aumailley, 1994). In particular, both LG2 and LG3 have been reported to have a binding site for integrin alpha3beta1 (Mizushima et al, 1997 and Shang et al, 2001). As a result, it is not surprising that the above described monoclonal antibodies directed to domains LG1-LG3 have been shown to display some activity on LN5 function and proposed for cancer treatment.
Although these antibodies have been generated a long time ago, none of them appears to have resulted in sufficiently satisfactory results to reach clinical trial. This is not surprising since the LG1-3 modules are (1) present in mature LN5, which is expressed in any epithelial basement membranes of normal tissues and (2) are involved in crucial integrin mediated cell adhesion mechanisms.
No binding site for integrin has been defined so far in LG4-LG5 domains of LN5 alpha3 chain. However, interactions with heparan sulphate proteoglycans in this region have been documented.
In particular, it has been shown that domain LG4 contains binding sites for syndecan-2 and syndecan-4 (Utani et al, 2001). In addition, domains LG4-LG5 (also abbreviated as LG4/5) have been shown to include a binding site for syndecan-1 (Okamoto et al., 2003; Bachy et al, 2008).
In addition, even though syndecan-1 binds to the LG4/5 domain when it is present in pre-LN5 or detached, it is not known whether the cleaved LG4/5 domains has a specific function.
WO 2005/073254 describes that LN5 alpha3 LG4/5 domains are important for squamous cell carcinoma (SCC) tumorigenesis. Indeed, this document first describes that transformed keratinocytes expressing only a truncated LN5 alpha3 chain gene lacking the LG4/5 domains fail to generate tumor invasion, while transformed keratinocytes with a normal LN5 alpha3 chain gene permit tumor invasion. This document then speculates on the usefulness of monoclonal antibodies to LN5 alpha3 LG4/5 domains for treating cancer. However, this is pure speculation since no such monoclonal antibody nor antigen to generate such antibody, is even prepared in this document.
Tran et al (2008) confirm the importance of LN5 alpha3 LG4/5 domains for squamous cell carcinoma (SCC) tumorigenesis. In addition, Tran et al show that treatment of mice suffering from SCC with a polyclonal antibody directed to LN5 alpha3 LG4/5 domains induced SCC tumor apoptosis, decreased SCC tumor proliferation, and impaired human SCC tumorigenesis in vivo without affecting normal tissue adhesion.
However, a polyclonal antibody is used, made of various specificities to distinct regions of LN5 alpha3 LG4/5 domains, that may target interactions with syndecan-1, 2, or 4, or even two or three of them. It thus cannot be derived from this article which region of LN5 alpha3 LG4/5 domains or which particular interaction(s) with syndecan-1, 2, and/or 4 should be targeted to reproduce anticancer activity.
In particular, while Tran et al show that heparin, as well as the anti-LN5 alpha3 LG4/5 polyclonal antibody, inhibit adhesion of transformed keratinocytes to LN5 alpha3 LG4/5 domains, it only speculates that interactions of LN5 alpha3 LG4/5 domains with syndecan-1, 2, and/or 4 may be implicated, with no indication in favour of syndecan-1.
In addition, using a polyclonal antibody, it is reasonable to think that the various antibody specificities target more than one of the interactions with syndecan-1, 2, and 4, and it is even not possible to conclude from this article that inhibition of only one of these interactions might be sufficient to obtain anticancer activity.
Two monoclonal antibodies directed to LN5 alpha3 LG4/5 domains have been described: clones 12C4 (Goldfinger et al., 1999) and D2-1 (Frank and Carter, 2004, Xia et al, 1996). However, no activity of these clones has been disclosed. Since both have been described several years ago, this clearly suggests that they have no activity on LN5 function, showing that not any region of LN5 alpha3 LG4/5 domains is suitable for obtaining an activity on LN5 function.
Thus, in view of the not well understood role of LG4/5 in cell adhesion and migration and in tumorigenesis, it is still not known from the prior art which region of the LG4/5 domains or which interaction of these domains with heparan sulphate proteoglycans (HSPG) syndecan-1, 2 and/or 4 should be targeted to obtain an anticancer activity. It is even not known if targeting of only one of these interactions might be sufficient to obtain anticancer activity.
In summary, it thus appears that various ligand molecules of LN5 (integrins alpha3beta1 and alpha3beta4, and HSPG syndecan-1, 2 and 4) are involved in its adhesion and migration functions, and may thus potentially be targeted for anticancer therapy using monoclonal antibodies.
However, the precise function of each of the interactions between LN5 and its ligands still remains to be elucidated, and it is currently not known which interaction should preferably be targeted for obtaining anticancer efficiency.
In addition, LN5 interacts with ligands on several of its three chains. For instance, there is a putative integrin binding site within the N-terminus of the gamma2 subunit (Decline and Rousselle, 2001). LN5 has also been reported to bind the extracellular domain of the transmembrane bullous pemphigoid antigen BP180 (Franzke et al., 2006). LN5 directly interacts with collagen type VII through the N-terminus of the beta3 laminin subunit and, to a lesser extent, the gamma2 LN subunit (Rousselle et al., 1997; Chen et al., 1999). In addition, the gamma2 subunit of LN5 has been shown to interact with type IV collagen, perlecan, and fibulin (Sasaki et al., 2001). LN5 also binds to other LN isoforms, LN 6 and 7 by the interaction of beta3 chain VI domain with the alpha3 chain short arm domain III of LN5 (Champliaud et al., 1996). This makes it even more difficult to choose a particular interaction and a particular chain domain.
There is thus a need for new anti-LG4/5 monoclonal antibodies with demonstrated anticancer activity.
The present invention provides monoclonal antibodies that inhibits the interaction between LN5 alpha3 chain LG4/5 domain and syndecan-1. In particular, the inventors generated three monoclonal antibodies, named 1H12, 3A11 and 15G5, which inhibit the interaction between LN5 alpha3 chain LG4/5 domain and syndecan-1, and which inhibits the growth of cells expressing pre-LN5 with the alpha3 chain LG4/5 domain, and particularly of colon carcinoma cells, breast carcinoma cells, and ovarian carcinoma cells.